Identification of the Pre–T-Cell Receptor Α Chain in Nonmammalian

Identiﬁcation of the pre–T-cell receptor α chain in nonmammalian vertebrates challenges the structure–function of the molecule

Philippe Smeltya, Céline Marchala, Romain Renarda, Ludivine Sinzelleb, Nicolas Polletb, Dominique Dunona, Thierry Jaffredoa, Jean-Yves Sirec, and Julien S. Fellaha,1

aUniversité Pierre et Marie Curie, Unité Mixte de Recherche-Centre National de la Recherche Scientifique 7622, 75252 Paris Cedex 05, France; bUniversité d’Evry Val d’Essonne, Genopole Centre National de la Recherche Scientifique, 91058 Evry, France; and cUniversité Pierre et Marie Curie, Unité Mixte de Recherche-Centre National de la Recherche Scientifique 7138, 75252 Paris Cedex 05, France

Edited by Philippa Marrack, National Jewish Health, Denver, CO, and approved October 13, 2010 (received for review July 15, 2010) In humans and mice, the early development of αβ T cells is controlled and the 3′ UTR. Recently, porcine PTCRA was reported to by the pre–T-cell receptor α chain (pTα) that is covalently associated possess an additional exon 1 designated “exon 1b,” the function with the T-cell receptor β (TCRβ) chain to form the pre–T-cell receptor of which is yet to be defined (7). However, despite the crucial (pre-TCR) at the thymocyte surface. Pre-TCR functions in a ligand- regularory role played by pTα in the control of αβ T-cell pro- independent manner through self-oligomerization mediated by pTα. duction, the presence of this protein in nonmammalian species Using in silico and gene synteny-based approaches, we identified has never been addressed. the pTα gene (PTCRA) in four sauropsid (three birds and one reptile) By combining in silico analyses and various molecular techni- genomes. We also identified 25 mammalian PTCRA sequences now ques, we describe PTCRA orthologs in sauropsids (birds and covering all mammalian lineages. Gene synteny around PTCRA is re- reptiles). We show that sauropsidian and mammalian PTCRA markably conserved in mammals but differences upstream of PTCRA share a common organization with conserved functional domains. in sauropsids suggest chromosomal rearrangements. PTCRA organi- In addition, the comparison of mammalian and sauropsidian gene zation is highly similar in sauropsids and mammals. However, com- synteny around PTCRA shows that this region has undergone parative analyses of the pTα functional domains indicate that sau- chromosomal rearrangements in a mammalian ancestor. More- ropsids, monotremes, marsupials, and lagomorphs display a short over, the pTα of sauropsidians and some mammals (monotremes, pTα cytoplasmic tail and lack most residues shown to be critical for marsupials, and lagomorphs) possess specific molecular features human and murine pre-TCR self-oligomerization. Chicken PTCRA distinct from human and mouse pTα. Taken together, our data transcripts similar to those in mammals were detected in immature lead us to propose a putative primary function for pTα in amniote double-negative and double-positive thymocytes. These findings ancestors and provide arguments for revisiting some of the give clues about the evolution of this key molecule in amniotes and mechanisms initially attributed to mammalian pTα signaling. α

suggest that the ancestral function of pT was exclusively to enable IMMUNOLOGY expression of the TCRβ chain at the thymocyte surface and to allow Results binding of pre-TCR to the CD3 complex. Together, our data provide Identification of Sauropsidian PTCRA. To target the genomic region arguments for revisiting the current model of pTα signaling. housing PTCRA, we used a strategy based on gene synteny conservation. Because PTCRA had been identified in only a few fi he early steps of intrathymic αβ T-cell production are con- mammalian species, we rst decided to screen all the available PTCRA SI Materials and trolled by the expression of the pre–T-cell receptor (pre-TCR) mammalian genomes for the presence of ( T Methods fi PTCRA through a mecanism called “TCRβ selection” (1). The pre-T cells ). We identi ed 25 mammalian sequences to- that harbor a productive T-cell receptor β (TCRβ) rearrangement gether with their gene environment (Fig. S1). We found that gene PTCRA express a pre-TCR composed of the TCRβ chain covalently linked synteny around was remarkably conserved in the mam- PTCRA to the invariant, nonrearranging pre-T–cell receptor α (pTα) chain malian lineage (Fig. 1). was located between ribosomal RPL7L1 CNPY3 associated with the CD3 complex (2). Signaling through the pre- protein L7-like 1 ( ) upstream and canopy 3 ( ) dwonstream. At least five genes upstream and nine genes down- TCR rescues developing T cells from programmed cell death and PTCRA induces further differentiation into immature T cells. Several im- stream of were conserved with the same respective order from human to platypus on both sides of the RPL7L1-PTCRA- portant functions have been attributed to the pre-TCR, including CNPY3 pre-T cell survival and proliferation, TCRβ allelic exclusion, T-cell segment (Fig. 1). Thus this gene synteny was present in the receptor α (TCRα) rearrangement, and induction of CD4 and last common mammalian ancestor and conserved for more than α 200 million years (My). CD8 expression (2). pT thus is an essential component of the pre- CNPY3 RPL7L1 TCR as shown in pTα gene (PTCRA)-deficient mice in which and therefore were considered the appro- αβ priate boundaries to look for PTCRA in the chicken genome (SI development of T cells is severely affected (1). Pre-TCR is Materials and Methods fi CNPY3 believed to function in a ligand-independent manner through self- ). We rst localized on chicken oligomerization mediated by the extracellular part of pTα and to activate signal transduction pathways (3). The cytoplasmic tail (CT) of pTα, in particular its proline-rich motif, was shown to be Author contributions: P.S., T.J., and J.S.F. designed research; P.S., C.M., R.R., L.S., J.-Y.S., and J.S.F. performed research; N.P., D.D., and J.-Y.S. contributed new reagents/analytic required for pre-TCR signaling (4). tools; P.S., L.S., N.P., D.D., T.J., J.-Y.S., and J.S.F. analyzed data; and T.J., J.-Y.S., and J.S.F. PTCRA was identified initially in human and mouse (5, 6). The wrote the paper. PTCRA transcription unit consists of four exons. Exon 1 encodes The authors declare no conflict of interest. the 5′ UTR, the leader peptide, and the first three amino acids of This article is a PNAS Direct Submission. the protein; exon 2 encodes the extracellular Ig-like domain; Data deposition: The chicken PTCRA sequences reported in this article have been depos- exon 3 encodes the connecting peptide that contains the cysteine ited in the GenBank database (accession no. HM630316). residue required for the interchain disulfide bond with the TCRβ 1To whom correspondence should be addressed. E-mail: [email protected]. chain; and exon 4, encodes the hydrophobic transmembrane This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. region, the long proline-rich CT involved in pre-TCR signaling, 1073/pnas.1010166107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1010166107 PNAS | November 16, 2010 | vol. 107 | no. 46 | 19991–19996 Downloaded by guest on September 25, 2021 were identified upstream chicken CNPY3 (Fig. 1). In human, these two genes map to chromosome 2, whereas PTCRA is found on chromosome 6. Because the putative PTCRA was not annotated in the chicken genome, we explored the genomic region located between CNPY3 and POLR1B with UniDPlot using mammalian PTCRA exon 2 as a template. A sequence recognized as the chicken PTCRA exon 2 was identified close to CNPY3, followed by the identification of exons 1 and 3. Exon 4 and the 3′ UTR were not found because a gap was present in the genomic region between PTCRA exon 3 and CNPY3 exon 2. We recovered the missing region using PCR on genomic DNA and obtained a sequence of approximatively 2 kb. Full-transcript sequences of chicken PTCRA then were obtained using RT-PCR and rapid amplification of cDNA ends (RACE)-PCR on RNA isolated from chicken thymus. Chicken PTCRA was composed of four exons (Fig. S2). Ex- ploring the intron 1 sequence did not yield any indication of the presence of exon 1b as found in some mammals (Fig. 2 and Fig. S1). The length of the first three exons was similar in mammalian and chicken PTCRA, but chicken exon 4 was shorter. In chicken, PTCRA introns were significantly shorter than their mammalian counterparts, a characteristic attributed to the high degree of genome compaction in this species. The longest PTCRA transcript encompassed 1,569 nt, including a short 5′ UTR (79 nt) and a large 3′ UTR (980 nt). A total of 510 nt codes the protein composed of 170 amino acids. The discovery of chicken PTCRA was instrumental in identifying its orthologs in two other bird genomes, zebrafinch and turkey, and in a lizard (Anolis carolinensis) genome. The sauropsidian sequences were validated using alignment with the chicken sequence. Lizard PTCRA exon 1 was not found in the genomic DNA because of a poorly assembled sequence at this locus. In sauropsids, gene synteny upstream of PTCRA differs from that described in mammals, confirming that a chromosomal rearrangement occurred in this genomic region after the divergence between the sauropsidian and mammalian lineages. Synteny immediately downstream of PTCRA was similar in the Fig. 1. Gene organization around PTCRA from representative osteichthyan four sauropsidian genomes, indicating that this region was stable genomes. In mammals (human, mouse, opossum, and platypus), gene synteny for more than 255 My; however, a synteny break was found be- is conserved with CNPY3 and RPL7L1 located respectively downstream and fi tween birds and lizard after peroxisomal biogenesis factor 6 upstream of PTCRA. In sauropsids (chicken, zebra nch, and lizard), gene syn- PEX6 teny is conserved downstream of PTCRA (CNPY3, GNMT,...)butisdifferent ( ) (Fig. 1). upstream (POLR1B, TTL, ...) than in mammals. In the frog, a gene synteny similartothatinsauropsidswasfoundonchromosome5,butPTCRA (marked Looking for PTCRA in Frog and Fish. We looked for Xenopus PTCRA “?”) could not be identified. In the zebrafish, genes and/or groups of genes in the frog genome with BLAST (SI Materials and Methods) using belonging to tetrapod gene syntenies were found distributed on at least three mammalian and sauropsidian PTCRA query sequences. Because chromosomes. The candidate region (13 kb) located upstream of CNPY3 on we did not find similar frog sequences, we proceeded using our chromosome 1 (double arrow) does not house PTCRA. Genes are depicted by gene synteny strategy. Genes syntenic to PTCRA were found in fi oriented pentagons. Of cial, full names of the genes annotated in the ge- amniote genomes but were located in different scaffolds [CNPY3 nomic regions and not mentioned in the text are as follows: ACSS1,acyl-CoA and glycine N-methyltransferase (GNMT) on scaffold 1241; TTL synthetase short-chain family member 1; ATL2, atlastin GTPase 2; CUL7,cullin and POLR1B on scaffold 614; and RPL7L1 on scaffold 1317]. We 7; DACHB,dachshundB;DDX48, eukaryotic translation initiation factor; PTCRA ENTPD6, ectonucleoside triphosphate diphosphohydrolase 6; FBLN7, fibulin 7; explored the regions likely to harbor (e.g., close to FBXO42, F-box protein 42; KIAA0240,noofficial name; KLC4,kinesinlight RPL7L1, POLR1B,orCNPY3) with UniDPlot using PTCRA exon chain 4; KLHDC3, kelch domain containing 3; MEA1, male-enhanced antigen 1; 2 sequences of lizard, birds, and mammals as templates, but we MRPL2, mitochondrial ribosomal protein L2; PPP2R5D, protein phosphatase 2, could not find similar frog sequences. This result suggested that regulatory subunit B′, delta isoform; PRPH2, peripherin 2 (retinal de- PTCRA is absent in the Xenopus genome, is missing in the avail- generation, slow); SLC22A7, solute carrier family 22 (organic anion trans- able genomic sequences, or is located elsewhere in the genome. porter), member 7; TBCC, tubulin folding cofactor C; TRPC5, transient receptor Therefore, we mapped putative syntenic genes using FISH of potential cation channel, subfamily C, member 5; TTBK1, tau tubulin kinase 1; chromosomes and labeled cDNA probes against various target UBR2, ubiquitin protein ligase E3 component n-recognin 2; VSX1, visual system SI Materials and Meth- homeobox 1; ZC3H6,zincfinger CCCH-type containing 6. genes or their contig regions (Fig. S3 and ods). Critical members of the gene synteny described in sauropsids (POLR1B, TTL, and CNPY3) were mapped on chromosome 5, chromosome 3 and subsequently identified several genes that strongly suggesting that Xenopus PTCRA could be located in the were syntenic downstream of CNPY3 in mammalian genomes. genomic region delimited by TTL and CNPY3 (Fig. 1). The contig RPL7L1, however, was found on chromosome 4, revealing that region housing RPL7L1 upstream of PTCRA in mammals was a chromosomal rearrangement in the vicinity of PTCRA had found on chromosome 8, indicating that the chromosomal rear- occurred in lineages leading to either modern birds or mammals. rangement occurred in a mammalian ancestor. Instead, polymerase (RNA) I polypeptide B (POLR1B) and tu- CNPY3 was identified in the five teleost fish genomes currently bulin tyrosine ligase (TTL), as well as several downstream genes, available (fugu, on scaffold 98; tetraodon, chromosome unplaced;

19992 | www.pnas.org/cgi/doi/10.1073/pnas.1010166107 Smelty et al. Downloaded by guest on September 25, 2021 Fig. 2. Amino acid comparison of pTα sequences in amniotes. The Homo sapiens pTα sequence is aligned with that of four sauropsids [chicken (Gallus gallus), turkey (Meleagris gallopavo), zebra finch (Taenopygia guttata), and lizard (Anolis carolinensis)] and six representative species of the main mammalian lineages [monotremes (platypus, Ornithorhynchus anatinus), marsupials (opossum, Monodelphis domestica), afrotherians (elephant, Loxodonta africana), leurasiatherians (cow, Bos taurus; and dog, Canis familiaris), and glires (mouse, Mus musculus)]. The signal peptide (shown within a box) is encoded by exon 1a. The highly variable C-terminal region encoded by exon 4 (shown in gray was aligned tentatively to highlight a few conserved residues in human, cow, dog, IMMUNOLOGY and elephant (Fig. S1). For convenience of presentation, amino acids (numbers are given within parentheses) were removed from the human, cow, and dog sequences. Residues identified as important in the mouse pTα sequence are indicated by gray columns. k, limits of exons; (.), residue identical to the human pTα residue; -, indel; #, unchanged residue; ?, unknown residue; *, Stop codon. Potential N-glycosylation sites are underlined. Amino acids not identified in the text: F, phenylalanine; I, isoleucine, M, methionine; Y, tyrosine. Cp, connecting peptide; Ct, cytoplasmic tail; Tm, transmembrane.

medaka, chromosome 18; zebrafish, chromosome 1; and stickle- (amino acids 36–143 in our alignment). The two cysteines (C64 back, group VII). However, a gene synteny similar to that de- and C124) required for the intrachain disulfide bridge also were scribed in tetrapods was not found, although several members conserved. The canonical tryptophan (W79) essential for stabi- of the synteny were found in various chromosomes (Fig. 1). We lization of the Ig-fold tertiary structure was present in all obtained no result when exploring with UniDPlot the DNA region sequences analyzed with the notable exception of lizard pTα. (13–20 kb) upstream of CNPY3 using amniote PTCRA sequences Most of the other conserved residues are known to be useful for as templates. We concluded that PTCRA either is absent from the the correct folding of the Ig-like domain, including the hydro- teleost fish genomes or is located in another genomic region and phobic amino acids valine (V66) and leucine (L88), the small was largely modified during evolution of the actinopterygian lin- hydrophilic amino acids proline (P44and P49), glycine (G101), and eage (450 My). threonine (T102), and aromatic tyrosine (Y93). The region encoded by exon 3 displayed a weaker conservation except for Comparison of Mammalian and Sauropsidian pTα. The 30 amino C153 that covalently links the TCRβ chain (5). acid sequences of mammalian pTα were aligned, taking the hu- Four charged amino acids [aspartic acid (D55) and arginine man sequence as a reference (Fig. S1). This alignment consisted (R57,R135, and R151)] located in the extracellular domain en- of 350 positions when considering the longest sequences and coded by exons 2 and 3 had been identified previously as crucial including the region encoded by exon 1b (20 residues in porcine residues for autonomous oligomerization and pre-TCR function pTα). A total of 41 amino acids was found unchanged. Exon 1b in murine pTα (Fig. 3) (8). Indeed, substitution of one of them was not present in basal mammalian lineages (monotremes and impeded self-oligomerization and pre-TCR ability to deliver marsupials), in most primates, or in some rodents, indicating that signals. It is remarkable that these residues were absent in sau- it was recruited in a placental ancestor (i.e., after divergence of ropsidian pTα.D55 was replaced with either a polar amino acid the monotreme and marsupial lineages) and then was lost in- [serine (S) or asparagine (N)] or a hydrophobic amino acid (al- dependently in several lineages. anine, A). R57 was replaced with either a bulky (glutamine, Q) or The sequences of the four sauropsidan pTα were aligned to an acidic (glutamic acid, E) residue; in birds, R135 was replaced seven pTα sequences representative of the main mammalian by another positively charged residue (histidine, H), whereas lineages (Fig. 2). Twenty-four positions remained unchanged. a tryptophan was found at this position in the lizard; and R151 These conserved residues were located mainly in the exon 2- was replaced with a negatively charged residue (E). Moreover, encoded region containing the extracellular Ig-like domain our alignment indicates that these positions also may vary in

Smelty et al. PNAS | November 16, 2010 | vol. 107 | no. 46 | 19993 Downloaded by guest on September 25, 2021 were observed in all positive samples. Independent sequencing analyses revealed that (i) the 500-bp product corresponded to the full-length PTCRA transcript; (ii) the 400-bp product con- tained two short PTCRA transcripts lacking the 5′ region of exon 2, resulting from two intraexonic splicing sites; and (iii) the largest transcript of 600 bp was composed of the four exons plus intron 1 (111 bp) sequence. This variant was generated from primary PTCRA RNA by missing the splice donor site of intron 1. However, although it was in a correct reading frame, the 600- bp PTCRA was unlikely to be translated, because a stop codon was generated immediately after the exon 1 splice site. The two shorter transcripts revealed a frameshift that changed the amino acid sequences leading to different and unknown proteins and generating a stop codon in exon 4 (Fig. S4). In situ hybridization was performed on thymus sections of E18 chicken embryos to localize PTCRA transcripts (Fig. S5). For comparison and because murine PTCRA expression is not docu- mented in the literature, we performed in situ hybridization using a mouse PTCRA probe on thymus sections of 6-wk-old mice. In both chicken and mouse, PTCRA transcripts were identiﬁed in cortical thymocytes. To identify more precisely the thymocyte subsets expressing PTCRA, we used ﬂow cytometry to sort the double-negative − − (DN, CD4 CD8 ), double-positive (DP, CD4+CD8+), and − − mature single-positive (SP, CD4+CD8 and CD4 CD8+) cell subpopulations. On average, these populations represented 15%, 80%, and 5% of the chicken thymocytes, respectively. RT-PCR analysis revealed that PTCRA transcripts were abundant in DN and DP cells but were not detected in SP thymocytes (Fig. 4B). These results strongly suggest that the expression of PTCRA is developmentally regulated in the chicken thymus, with a high level of expression in the most immature thymocytes, as de- Fig. 3. Alignment of mammalian and sauropsidian sequences in two pTα scribed in the mouse (9). regions in which some amino acids (shown in gray) were found to play an important role. Symbols are as in Fig. 2. Discussion Phylogenetic Analysis of Amniote PTCRA. The discovery of PTCRA

135 in sauropsidians moves back the origin of this gene at least to the some mammalian pTα (e.g., R replaced by a glutamic acid, amniote ancestors, suggesting that the major mechanisms that a tryptophan, or a proline). control αβ T cell development were conserved for more than 310 The 5′ sequence of exon 4 encodes the transmembrane region My. Gene synteny around PTCRA was stable among mammals of pTα that contains several hydrophobic amino acids and the but was subjected to at least two rearrangements during amniote 174 179 two canonical amino acids arginine (R ) and lysine (K ). evolution: One, after the divergence of sauropsids and mammals, These conserved residues are believed to be involved in charged modified the whole chromosomal segment upstream of PTCRA; interactions with the transmembrane domain of a component of the other, after the divergence of reptiles and birds, occurred at the CD3 complex (5). In all pTα sequences analyzed, these basic a higher distance from PTCRA. The formal presence of PTCRA and positively charged residues are always separated by four in a tetrapod ancestor is yet to be proven, but the synteny we nonconserved amino acids (Fig. 2). evidenced in Xenopus is in keeping with the existence of PTCRA 188 The C-terminal region of pTα, starting at T and encoded by in this region. PTCRA was not identified in teleost fish, sug- the 3′ region of exon 4, consists in the CT of the protein pre- gesting that it either is absent or is located in another chromo- viously shown to be critical for pTα signaling. In mammalian somal region. If pTα is not present in fish, the most probable pTα, this region was found to be highly variable both in length hypothesis is that an early expression of the αβ TCR in DN cells − − and amino-acid composition and was difficult to align with the could contribute to production of DP cells as observed in Tcrd / − − exception of primates and small regions in cetartiodactyls (Fig. 2 Ptcra / mice in which an early expressed αβ TCR subsitutes for and Fig. S1). The shortest CTs were found in lagomorphs (pika pre-TCR (2). and rabbit, with six and eight amino acids, respectively); the The evolutionary analysis of the PTCRA structure in amniotes largest were encountered in primates (110 amino acids) and clearly indicates an ancestral structure typically composed of four cetartiodactyls, in particular cow (140 amino acids) and dolphin exons. The fifth exon, 1b, was recruited in a placental ancestor and (129 amino acids). The CTs of mammalian pTα possess nu- then was lost independently in several mammalian lineages, a his- merous prolines believed to play a role in pre-TCR signaling (4), tory that does not support an important function for the pTα re- but CTs of lagomorphs, marsupials and monotremes do not. In gion encoded by this exon. The comparison between mammalian addition, all sauropsidian pTα possess a short CT with only seven and sauropsidian pTα sequences revealed that some positions and amino acids and no proline. domains shown to be crucial for pTα function in mammals are not conserved through evolution, thus challenging the complex func- Expression of Chicken PTCRA. Using RT-PCR experiments, we tional model of pTα previously proposed in human and mouse. detected PTCRA transcripts in thymus, spleen, and bone marrow of chicken embryos at 10 d (E10), 14 d (E14), and 18 d (E18) Comparative Studies of Functional Domains in Amniote pTα. CT. A (Fig.4 A). No amplification was obtained in liver and gut sam- particular feature of the sauropsidian pTα is the lack of a long ples. Three bands of 600 bp, 500 bp, and 400 bp, respectively, CT. This region was shown to be crucial for the pre-TCR func-

19994 | www.pnas.org/cgi/doi/10.1073/pnas.1010166107 Smelty et al. Downloaded by guest on September 25, 2021 Fig. 4. Expression of PTCRA transcripts. (A) PTCRA expression in hematopoietic tissues of E18, E14, and E10 chicken embryos was analyzed by RT-PCR. Three bands of 600 bp, 500 bp, and 400 bp, respectively, were identiﬁed in all of the stages. Bm, bone marrow; RT-, control PCR in which reverse transcriptase was omitted during ﬁrst-strand synthesis; Sp, spleen; Th, thymus. S17 primers were used to assess the amount of cDNA samples used for PCR experiments. (B)(Left) Flow cytometric isolation of E18 chicken thymocyte subsets analyzed in Right.(Right) RT-PCR analysis of PTCRA expression on thymocyte subsets. PTCRA transcripts were detected in DN and DP thymocytes. S17 primers were used to assess the amount of cDNA samples used for PCR experiments.

tion in mice and humans (2, 4). In particular, the proline-rich mammals. This expression would strengthen the pre-TCR signal sequences were proposed to interact with the SRC homology and compensate for the absence of costimulating signal mediated (SH3) domain of Rous sarcoma oncogene (Src) kinases and with by the pTα CT. Thus, the critical biological functions previously members of the cytoplasmic cas ligand with multiple SH3 attributed to the pTα CT appear to have been overestimated and domains/Cbl interacting protein of 85 kDa adaptor family (10). need to be reexamined thoroughly. Transgenic mice in which pTα has been replaced by a mutant The TCRβ chain structure, and particularly most residues in- form lacking the entire cytoplasmic domain display defects in volved in the interaction with the TCRα chain and the compo- pre-TCR–induced proliferation and survival that result in re- nents of the CD3 complex, is well conserved from cartilaginous duced numbers of thymocytes, inhibition of the DN–DP transi- ﬁsh to mammals. However, nonmammalian TCRβ chains lack tion, and defective TCRβ selection (4). The comparison between a hydropholic loop of about 14 residues, an important structure of mammalian and sauropsidian pTα raises questions about the mammalian Cβ regions (14). In mammals, this loop is exposed to critical role of the CT domain. Indeed, the length and amino- solvent and may interact with a component of the CD3 complex.

acid composition of the CT is highly variable among mammals, The absence of this loop suggests that in nonmammals the Cβ Ig IMMUNOLOGY and some (e.g., lagomorphs, marsupials, and monotremes) lack fold is associated with the cell surface more closely than in the proline-rich region, thus raising questions about the absolute mammals, with probable consequences for the spatial configura- requirement of this motif for proper pTα signaling and function. tion of the αβ TCR and its association with CD3 molecules. The In sauropsidian pTα the CT is short (seven amino acids) and same considerations might apply to pTα/TCRβ/CD3 interactions does not contain prolines. Therefore, αβ T-cell development can in nonmammals, probably with some consequences on the pre- occur in the absence of a long, proline-rich pTα CT. Moreover, TCR signaling capacity. Thus, the absence of the hydropholic the mechanisms involved in diversification of the αβ TCR rep- loop on nonmammalian TCRβ chains could be related to the ertoire are similar in birds and mammals, suggesting that pre- reduced length of the pTα CT. In sauropsids, the CD3 complex TCR are equally efficient in generating αβ T cells in these two probably is crucial for pre-TCR signaling, because the charged lineages despite the absence of a functional pTα CT in birds (11). residues involved in the interaction with the CD3 complex are In mammals possessing a long pTα CT (e.g., rodents, primates, conserved. Immunofluorescence experiments in chicken thymo- and cetartiodactyls) interacting with multiple signaling mole- cytes have shown that the CD3 complex is expressed at the sur- cules, this feature could have been selected during evolution to face of the immature DN thymocytes (Fig. S6). optimize the propagation of the pre-TCR signal. It also has been Extracellular domain. Mutagenesis analyses revealed that the posi- postulated that the long CT could favor interactions with pro- tively charged amino acids (D55,R57,R135,andR151) located in teins located in lipid rafts, allowing the appropriate pre-TCR the extracellular domain of human and murine pTα are crucial localization required for autonomous signaling (4). The short CT for oligomer formation (10), a mechanism essential for ligand- identified in sauropsids and in some mammals does not allow independent signaling by pre-TCR. In the mouse, experimental generalizing this hypothesis. Our findings suggest that the long replacement of R57 by a glutamine completely abolishes the ability pTα CT could function by costimulating rather than initiating of pTα to support pre-TCR signaling, and substitution of D55 by an pre-TCR signaling, as initially proposed (12). alanine abrogates pre-TCR activity. These residues are not con- Another function attributed to pTα CT is directly fine-tuning served in sauropsids or in some mammals. In particular, R57 often constitutive endocytosis and degradation of the pre-TCR/CD3 is replaced by a glutamine, and in the zebrafinch D55 is replaced by complex and the level of expression of pre-TCR at the cell sur- an alanine, calling into question the critical function of these face (13). This mechanism is thought to avoid sustained ligand- amino acids. How could these apparently contradicting results be independent signaling through a potentially oncogenic cell- interpreted? (i) Mouse, human, and a few other mammals might growth receptor. In sauropsids, the absence of a long CT could represent a particular situation in which positions 55, 57, 135, and result in an increased expression of the pre-TCR/CD3 complex 151 were selected for pre-TCR oligomerization. (ii) Stretches of at the plasma membrane to levels similar to those of αβ TCR, as specific residues, rather than precisely located important amino suggested in the mouse bearing an experimental deletion of pTα acids, might allow oligomerization. This situation appears to be CT (4). Therefore, we suggest that sauropsidians display an en- the case in birds where two arginines are found in the neighboring hanced expression of pre-TCR at the cell surface compared with positions 58 and 59. (iii) Pre-TCR could deliver its signal in the

Smelty et al. PNAS | November 16, 2010 | vol. 107 | no. 46 | 19995 Downloaded by guest on September 25, 2021 absence of oligomerization in sauropsids and in basal mammalian tional differences between the two pTα isoforms (15). In chicken, lineages. Further investigation is required to clarify this situation. the physiological function of the short PTCRA transcripts was difficult to understand, and whether these transcripts compete Comparative Analysis of PTCRA Expression. In chicken, T-cell pro- with the large one and regulate the amount of pTα remains to genitors enter the thymus in three successive waves that start at be answered. embryonic day (E)6, E12, and around E18 (just before hatching), Our study demonstrates that pTα, and therefore the pre-TCR respectively. Our RT-PCR results indicated that each wave of receptor, exists in sauropsidians and already was present in the thymocyte progenitors generates T-cell precursors that express last common amniote ancestor, more than 310 Mya. Comparative PTCRA PTCRA . In the mouse, expression increases from DN1 structure–function analyses of the pTα sequences revealed that fi to DN4 thymocyte subsets identi ed by the expression of CD25 sauropsidian and some mammalian pTα display a short CT and and CD44 (9). At the DP stage, PTCRA expression declines α α lack residues known to be required for pT signaling and self- concomitantly with the initiation of TCR locus rearrangement. oligomerization in humans and mice. These features do not result In the chicken thymus, similar DN, DP, and mature SP thymo- in any apparent change of thymocyte maturation and repertoire cyte populations have been characterized clearly (11). Unfor- diversity. This result suggests that the involvement of pTα in pre- tunately, because of the lack of specific antibodies against TCR signaling might be less crucial than previously believed from chicken CD25, a more accurate identification of the different studies in the mouse. In many species, pTα might act only as DN subsets could not be achieved. However, our results in pu- a surrogate TCRα chain to allow the expression of TCRβ linked to rified thymocyte subsets clearly indicate that PTCRA transcription occurs in immature DN and DP thymocytes but not in the the CD3 complex forming the pre-TCR complex on immature mature SP subset. These results are consistent with those thymocyte membranes. This function could to be similar to the PTCRA function attributed to the surrogate light chain that links the Ig obtained by in situ hybridization in which transcripts are μ – found exclusively in the cortical region of the thymus, where the -heavy chain to form the pre-BCR on the pre B-cell surface. early steps of thymocyte differentiation occur. Thus, during T- Materials and Methods cell development in chicken, the pattern of PTCRA expression is similar to that described in mammals. However, chicken PTCRA Database Search. Mammalian, sauropsidian, and nonamniote PTCRA or genes syntenic to PTCRA (CNPY3 and RPL7L1) were searched in genomic databases expression is not restricted to the thymus, because transcripts fi (National Center for Biotechnology Information and Ensembl) with BLAST were identi ed in spleen and bone marrow, suggesting the pre- and UniDPlot programs (SI Materials and Methods). sence of T-cell progenitors in these tissues, as also reported in PTCRA fi humans and mice (9). Three transcripts were identi ed PCR Experiments. PCR was performed using 1 μg of chicken genomic DNA. We in the chicken thymus, but only the transcript of 500 bp encodes obtained 5′ and 3′ UTRs of chicken PTCRA by rapid amplification of cDNA for the complete pTα. The other short transcripts lacking part of ends (RACE)-PCR using total RNA extracted from E18 thymus and the com- exon 2 encode proteins different from pTα because of a frame- mercial SMART–RACE cDNA amplification kit (Clontech). Tissue expression shift in their sequences. A short transcript lacking exon 2 was was analyzed by RT-PCR on total RNA from E10, E14, and E18 chicken thy- described previously in human and mouse PTCRA (2). This mus, spleen, bone marrow, gut, and liver. Primers used in PCR experiments transcript encodes a pTα isoform lacking the extracellular Ig-like are listed in SI Materials and Methods. domain but containing the cysteine involved in the covalent interaction with TCRβ chain. Although the function of this short Cell Sorting. The three thymocyte subsets (DN, DP, and SP) were sorted using fi isoform was not clearly elucidated, it was postulated that it could speci c anti-chicken CD4 and anti-chicken CD8 (Southern Biotechnology compete with the full-length pTα for TCRβ binding, thus limiting Associates) (SI Materials and Methods). Analysis and sorting were performed using an Influx 500 cell sorter (BD Biosciences). the amount of complete pre-TCR at the thymocyte surface. In chicken thymus and in sorted thymocyte subsets, the expression ACKNOWLEDGMENTS. We thank Drs. C. Durand, S. Kaveri, and F. Dieterlen of the short transcripts predominated during all the stages ana- for critical reading of the manuscript; A. Munier and N. Boggetto for flow lyzed. This result is in constrast with the situation in human and cytometry and cell sorting; S. Gournet for the illustrations; and A. Dady, R. mouse thymus, where the expression of the complete transcript Gautier, and G. Villain for technical advice. This work was supported by the Université Pierre et Marie Curie and the Centre National de la Recherche was 10-fold more abundant than the short transcript. In human Scientifique (Unité Mixte de Recherche 7622). P.S. is the recipient of a fellow- thymocytes, the expression of the two transcripts was indepen- ship from the Ministry of Education and Scientific Research and the Fonda- dently regulated during intrathymic development, reflecting function pour la Recherche Médicale.

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